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Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia

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Abstract

Loading of Ca2+-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca2+ ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca2+ indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca2+ distribution increased in the three apertures of pollen after hydration and showed a Ca2+ gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca2+ activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pHi regulated pollen tube growth.

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Abbreviations

BCECF-AM:

3′-O-Acetyl-2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein diacetoxymethyl ester

CLSM:

Confocal laser scanning microscopy

[Ca2+]cyt :

Cytosolic-free calcium

dd water:

Double distilled water

ECFP:

Enhanced cyan fluorescent protein

EGTA:

Ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

EYFP:

Enhanced yellow fluorescent protein

pHi :

Intracellular pH

SSR:

Shortest significance ranges

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Acknowledgments

We thank Professor Megumi Iwano of Nara Institute of Science and Technology (Japan) who supplied transgenic seed that expressed the YC3.1 gene in pollen grains and papilla cells. We also thank Professor Deng Shaoping in ZheJiang GongShang University who let us freely use the confocal instrument in his laboratory. We thank all the scientists who helped to improve our manuscript. We are indebted to the two anonymous reviewers, whose insightful comments resulted in a much improved final version of the manuscript. This work was supported by the National Natural Science Foundation of China (31071759) and the Scientific Research Fund of Huaiyin Institute of Technology (No: HG0606).

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Authors and Affiliations

  1. College of Horticulture, Nanjing Agricultural University, No.1. WeiGang, Nanjing, Jiangsu, China

    Haiyong Qu, Xueting Jiang & Shaoling Zhang

  2. Zhejiang Academy of Agricultural Sciences, No.198. Shiqiao, Hangzhou, Zhejiang, China

    Zebin Shi

  3. Huaiyin Institute of Technology, No.1. Meicheng Road, Huaian, Jiangsu, China

    Lianmei Liu

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  1. Haiyong Qu
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  2. Xueting Jiang
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  5. Shaoling Zhang
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Corresponding author

Correspondence to Shaoling Zhang.

Electronic supplementary material

Below is the link to the electronic supplementary material.10265_2011_440_MOESM2_ESM.doc

There are three phases of movie of the same pollen tube. Pollen were cultured in dye-free medium for 2 h in darkness after loading BCECF-AM at high temperature (37°C) for 30 min and then observed with CLSM. Confocal images were collected every 10 min for 3 h. We adjusted field of vision of CLSM every hour, because of movement from pollen tube growth. The first movie (pH 1.mpg) is images collected during the first 0–50 min (mpg 525 kb)

The second movie (pH 2.mpg) is images collected during the middle 60–130 min (mpg 829 kb)

The third movie (pH 3.mpg) is images collected in the last 50 min. These movies reveal the change of pH dynamics at of pollen tube tips during their growth (mpg 423 kb)

Supplementary material 1 (DOC 103 kb)

Supplementary material 2 (DOC 10,543 kb)

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Qu, H., Jiang, X., Shi, Z. et al. Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia . J Plant Res 125, 185–195 (2012). https://doi.org/10.1007/s10265-011-0440-z

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  • DOI: https://doi.org/10.1007/s10265-011-0440-z

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